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Preliminary exploration on pretreatment with metal chlorides and enzymatic hydrolysis of bagasse
b i o m a s s a n d b i o e n e r g y 7 1 ( 2 0 1 4 ) 3 1 1 e3 1 7
Available online at www.sciencedirect.com
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Preliminary exploration on pretreatment with metal chlorides and enzymatic hydrolysis of bagasse Liheng Chen, Rong Chen, Shiyu Fu* State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
article info
abstract
Article history:
Converting biomass to fermentable sugar is the critical step in the biomass refinery.
Received 26 May 2014
Moreover, pretreatment of biomass plays an important role in improving the conversion of
Received in revised form
biomass to sugar. In this study, sugarcane bagasse was pretreated by metal chloride Lewis
24 September 2014
acids (0.1 mol L3 CrCl3, FeCl3, FeCl2, ZnCl2 and AlCl3 solution) for cellulase hydrolysis. The
Accepted 25 September 2014
effects of pretreatments on the yield, chemical components, and sequential cellulase hy-
Available online 16 October 2014
drolysis of pretreated bagasse were investigated. The results indicated that metal chlorides with different pKa values could efficiently remove the hemicellulose in bagasse during
Keywords:
pretreatment. Furthermore, an inhibition factor (IF) quantitatively reflecting difficulty of
Sugarcane bagasse
cellulase hydrolysis was proposed. The low IF means the facile cellulase hydrolysis. The IF
pKa value
of Fe (III)-pretreated bagasse could decrease to 1.35. In this case, the enzymatic digestibility
Metal chloride Lewis acid
of bagasse approached to 100%.
Pretreatment
© 2014 Elsevier Ltd. All rights reserved.
Cellulase hydrolysis
1.
Introduction
To pursue alternatives to fossil energies is an indispensable trend, as the fossil energies suffer from the limited source and deteriorating environment [1,2]. Biofuels such as bio-alcohols, regarded as environmentally friendly alternatives, have been exploited extensively [3]. Lignocellulosic bioconversion is a competitive way to produce bioethanol because of its mild operating condition and the absence of by-products [4]. However, lignocellulose, feedstock of the process, has a rigid matrix structure comprised of cellulose, hemicellulose and lignin [5]. The presence of lignin and hemicellulose makes lignocellulosic bioconversion, especially the enzymatic hydrolysis of cellulose, hard to be implemented [6,7]. Generally, * Corresponding author. Tel.: þ86 20 87113940; fax: þ86 20 22236078. E-mail address: [email protected] (S. Fu). http://dx.doi.org/10.1016/j.biombioe.2014.09.026 0961-9534/© 2014 Elsevier Ltd. All rights reserved.
the factors that have been identified to affect the hydrolysis of cellulose include porosity (accessible surface area) of the waste materials, cellulose fiber crystallinity, and lignin and hemicellulose content [8]. The removal of lignin and hemicellulose from biomass, reduction of cellulose crystallinity and augment of porosity in pretreatment processes can significantly improve the enzymatic hydrolysis of biomass. Hence, pretreatment prior to enzymatic hydrolysis is imperative for lignocellulosic feedstock. Variety of biomass pretreatments have been reported to promote the lignocellulosic bioconversion and cut down the total costs. The pretreatment is usually performed by steam explosion, liquid hot water, dilute acid, inorganic salt, lime and ammonia [9]. Each pretreatment method has its own advantages and disadvantages
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for specific lignocellulosic feedstock [9]. In all cases, pretreatment, which could effectively remove hemicellulose in biomass, is crucial to enzymatic hydrolysis of lignocellulose. However, a complicated factor is that different biomass applied to the same pretreatment process will present different patterns of hemicellulose or lignin removal and of enzymatic digestibility, even under the same conditions [10]. In this study, sugarcane bagasse was pretreated by metal chloride Lewis acids for cellulase hydrolysis. With regard to the feedstock, sugarcane bagasse is a typical agricultural residue, derived from sugarcane after juice extraction [11]. In order to make full use of bagasse, it is essential to pursue a promising route such as bioconversion [12]. For pretreatment methods, metal chlorides have been applied to some biomass species, such as corn stover and waste fiber, which were reported to have a great effect on the sequent enzymatic hydrolysis of biomass [13e15]. Metal chlorides are particularly attractive as lignocellulose pretreatment agents, because they are less corrosive than inorganic acids and can be recycled [13]. Moreover, these metal chlorides were performed on the conversion of bagasse to sugar could not only expand the use of bagasse, but explore the suitability of this pretreatment process. Since high efficiency and economic feasibility of pretreatment is vital for large scale commercialization of biofuels and value added bio-chemicals, the kinetics of pretreatment for lignocellulosic bioconversion need to study to get an optimal condition. However, little information about the kinetics of enzymatic hydrolysis of lignocellulose pretreated by metal chlorides was reported except the effects of metal chlorides on biomass pretreatment [16]. In the recent years, the MichaeliseMenten equation has been widely used to study kinetics of enzymes performed on various substrates [17,18]. Nevertheless, it is not suitable for cellulase hydrolysis of lignocellulosic biomass, owing to the complexity of the enzymatic hydrolysis of biomass which is heterogeneous insoluble substrates [17]. Herein, we would investigate the effects of metal chloride Lewis acids pretreatment on the bagasse enzymatic hydrolysis, as well as explore the kinetics for further understanding the mechanism of these pretreatment.
2.
Materials and methods
2.1.
Materials
The feedstock, sugarcane bagasse, was obtained from Guangxi Guitang Group, Guangxi province, China. The sugarcane (Saccharum spp. hybrids) used in this sugar mill was cultivated in Guigang (Guangxi province, China) and harvested at an age of (10e11) months by harvester. The sugarcane was pressed through rollers to abstract the juice. The solid residues, known as bagasse, were air-dried and used in this study after depithing. They were milled by micro plant grinding machine (FZ102, Tianjin Taisite Instrument Co. China), collected through 10-mesh screen and then air-dried for further use. The mass fractions of glucan, xylan, lignin and ash in air-dried sugarcane bagasse were (36.02 ± 0.31) %, (19.41 ± 0.07) %, (24.20 ± 1.60) % and (0.55 ± 0.03) %,
respectively. Chemicals used in this paper were of analytical reagent grade. All experiments were performed in duplicate under the same conditions, and average values were reported.
2.2.
Pretreatment
Pretreatment was performed in a high pressure reactor (4530 series, Parr Co., US) with a total volume of 1 L. The bagasse of 30 g dry weight was loaded in the reactor, and mixed with the metal chloride solution. The total volume of liquid was 300 cm3. The initial concentration of metal chloride (CrCl3, FeCl2, FeCl3, ZnCl2 and AlCl3) for the pretreatment was 0.1 mol L1. In addition, dilute sulfuric acid pretreatment was carried out in this study as a control method to compare with metal chloride pretreatment. The sulfuric acid (about 0.4 g) was added until the pH value equaled to solution of ferric chloride pretreatment. After complete agitation, the pH value of solution was detected by DELTA 320 pH Meter. The reactants were initially at room temperature, and then heated to 170 C for 30 min. The reactor was immediately removed from the heating jacket and allowed to cool down when the pretreatment was finished. The pretreated bagasse was then separated by filtration. The liquid part of pretreatment was analyzed by pH meter, ion chromatography (IC) and highpressure liquid chromatography (HPLC) to determine pH values, the concentration of glucose, xylose, 5hydroxymethylfurfural (HMF) and furfural, respectively. The solid portion rinsed with deionic water was used for the cellulase hydrolysis. Meanwhile, composites in the pretreated solid were analyzed.
2.3.
Enzymatic hydrolysis
Pretreated bagasse of 1 g dry weight was hydrolyzed in the 100 mL flask by cellulase (Celluclast 1.5 L) with 20 Filter Paper Unit (FPU) per 1 g substrate and b-glucosidase (Novozyme 188) with 25 cellobiase units (CBU) per 1 g substrate. The cellulase hydrolysis of reaction mixture (50 cm3) was carried out in a HAc/NaAc buffer (0.05 mol L1, pH ¼ 4.8) on a rotary shaker (50 C, 2.5 Hz). Aliquots of 0.1 cm3 were taken at different time points, kept in boiling water for 1 min to inactivate the cellulase, and then centrifuged to remove water-insoluble solids. The supernatant of samples was analyzed by the glucose oxidaseperoxidase method (GOPM) for glucose content [19]. The cellulose enzymatic digestibility (CED) of pretreated bagasse was described as follows: CED ¼
2.4.
0:9 Glucose Mass 100% Substrate Mass Mass Fractrion of Glucan
(1)
Analysis methods
The components of treated fibers were determined according to the National Renewable Energy Laboratory (NREL, Golden, CO) analytical methods for biomass [20]. All liquid fractions for analysis by IC were diluted appropriately with the ultrapure water and then filtered by a 0.22 mm filter. The concentration of glucose and xylose were determined by IC system (Dionex ICS-3000) with a CarboPac PA20 column at 30 C. The quantification of HMF and furfural in the pretreatment
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solution were carried out by HPLC system with C18 column at 30 C based on a diode array detector (DAD). The eluents containing methanol (30%, volume basis) and water (70%, volume basis) flew at a rate of 1.0 cm3 min1.
3.
Results and discussion
3.1. Hydrolysis reaction of metal chlorides in water during pretreatment Generally, the acid strength plays an important role in acid pretreatments of lignocellulose [21]. Among metal chlorides pretreatment, the pKa values based on the acid dissociation constant were determined to indicate their acid strength [16]. Table 1 here e summarizes the pKa values of different metal chlorides in water at 25 C [22]. The lower pKa value indicated the more robust acidic character of the metal ion in the solution. The data in Table 1 showed that Fe (III), which pKa value is 2.73, forms stronger acids in aqueous solution than other cations. The pH values were measured for all the solutions (metal chloride solution mixed with bagasse) prior to pretreatment and after pretreatment. Fig. 1 here e was described as the relationship between initial and final pH values of liquid portion during different pretreatments. The red dash line (in web version) indicated the equivalent value of initial and final pH. When the data were located upon the dash line, it means that the final pH value was larger than the initial one. On the contrary, the final pH value would be less than the initial one. As we see from Fig. 1, pH values of liquid fraction in all pretreatment systems except dilute sulfuric acid pretreatment decreased. These results demonstrated that metal chlorides underwent hydrolysis to release more hydrogen ions during pretreatment as weak electrolyte. Hence, the ongoing releasing hydrogen ions and the probable produce of acetic acid from hemicellulose cause inevitably the decrease of the pH values of liquor after metal chloride pretreatments. Nevertheless, the sulfuric acid is a stronger electrolyte than acetic acid, which is the weak one. During the dilute sulfuric acid pretreatment, the sulfuric acid hydrolyzed entirely firstly. The exhaustion of hydrogen ions was much more than the produce from acetic acid hydrolysis, which resulted in the pH value rising from 1.6 to 3.82. Compared with the inorganic acids of strong electrolyte, metal chloride Lewis acids could provide steadier reactive environment and be less corrosive as well.
3.2. Component change of sugarcane bagasse after different metal chloride pretreatment It is essential to detect the component change of lignocellulosic material during pretreatment. The main components
Table 1 e The pKa value at 25 C. Methods
25 C pKa
Fe(III)
Cr(III)
Al(III)
Fe(II)
Zn(II)
2.74
4.01
5.03
6.8
6.31
Fig. 1 e Initial and final pH values of liquid portion during different pretreatment systems.
(glucan, xylan and Klason lignin) of sugarcane bagasse (SB) pretreated by different metal chlorides and dilute sulfuric acid were summarized in e Table 2 here. In general, the mass fractions of glucan and lignin in pretreated bagasse increased in comparison to those in raw bagasse, and the mass fraction of xylan in bagasse after pretreatment decreased significantly, which made the bagasse suffer from numeric mass loss. For instance, the largest remained solid mass of bagasse pretreated by dilute sulfuric acid was only 74.5%, and the least remained solid mass could reduce to 54.0% after chrome chloride pretreatment. The reason for mass loss of bagasse was mainly that the carbohydrates in bagasse degraded during pretreatment in the acidic environment. The extent of carbohydrate degradation could be also detected via analysis of pretreatment effluent. Table 3 here e outlined the components of pretreatment effluent. Numerous monosaccharides such as xylose and glucose were released, even further degraded to furfural and 5-hydroxymethylfurfural (HMF) as shown in Table 3. The greatest concentration (13.5 g L1 and 7.55 g L1) of xylose and glucose in pretreatment effluent could be attained during Cr (III) pretreatment. However, pretreatment by Fe (III) could produce more furfural, which reached 5.11 g L1, greater than that by Cr (III). It suggested that xylose during Fe (III) pretreatment was conversed to furfural more easily.
Table 2 e Mass fractions of components in pretreated sugarcane bagasse (%). Sample
Solid remain
Raw SB Fe(III) Cr(III) Al(III) Fe(II) Zn(II) H2SO4
e 58.0 54.0 70.4 71.3 72.2 74.5
Glucan 36.0 46.6 43.7 50.2 51.3 50.9 49.9
± 0.3 ± 2.3 ± 1.6 ± 1.4 ± 1.3 ± 0.9 ± 0.7
Xylan 19.4 ± 2.15 ± 3.33 ± 9.06 ± 12.8 ± 14.7 ± 16.7 ±
0.1 0.78 0.02 0.9 0.7 1.2 0.5
Lignin 24.2 41.3 44.7 35.0 33.3 33.7 32.3
± 1.6 ± 1.3 ± 0.6 ± 1.1 ± 0.6 ± 1.2 ± 0.9
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Table 3 e Components of pretreatment effluent. Concentration (g L1)
Method Xylose Fe(III) Cr(III) Al(III) Fe(II) Zn(II) H2SO4
11.3 ± 13.5 ± 10.2 ± 6.31 ± 3.61 ± 3.40 ±
0.3 0.1 0.1 0.08 0.23 0.10
Glucose
Furfural
6.77 ± 0.12 7.55 ± 0.24 0.736 ± 0.031 1.25 ± 0.04 0.357 ± 0.009 0.325 ± 0.013
Fig. 2a and b here e illustrated the existing forms of xylan and glucan in bagasse after different pretreatment and its relationship with pKa values of metal chlorides. The carbohydrates such as xylan and glucan underwent various extent degradations during pretreatment by metal chlorides of different pKa values. Especially, the xylan was more readily to
5.11 1.88 1.70 3.72 3.46 0.870
± 0.15 ± 0.47 ± 0.10 ± 0.10 ± 0.05 ± 0.002
HMF 0.752 0.667 0.816 0.268 0.252 0.069
± 0.030 ± 0.003 ± 0.058 ± 0.007 ± 0.005 ± 0.022
be destroyed among the pretreatment systems. In general, the xylan remained in bagasse pretreated by metal chlorides improved with the increase of pKa values, but less than that by dilute sulfuric acid pretreatment (Fig. 2a). The capability of pretreatment systems about degrading xylan was as follow: Fe (III) > Cr (III) > Al (III) > Fe (II) > Zn (II) > H2SO4 (Fig. 2a). The xylan was almost removed entirely in the Fe (III) and Cr (III) pretreated bagasse, their removal rates could reach 93.6% and 90.7%, respectively. Furthermore, glucan in these two pretreated bagasse suffered from some extent degradation, where the recovery of glucan decreased to 75.1% and 65.6%, respectively (Fig. 2b). However, other pretreatment systems could hardly degrade glucan. These results might result from the lower pKa values of Fe (III) and Cr (III) than others. In addition, the recovery of lignin in pretreated bagasse was approximately 100%, which indicated that all these pretreatment systems did not have delignification effect.
3.3. The pKa values of metal chlorides on the cellulose enzymatic digestibility of pretreated bagasse The pKa values of metal chlorides had been proved to have great influence on the bagasse pretreatment by metal chlorides. In this part, we tried to find out how the pKa value of metal chloride Lewis acids affected the sequential enzymatic hydrolysis of pretreated bagasse. Fig. 3 here e depicted the
Fig. 2 e Existing forms of xylan (a) and glucan (b) in bagasse after being pretreated with metal chloride and dilute sulfuric acid. X1 and G1 are the remaining xylan and glucan in the residue; X2 and G2 are monomeric xylose and glucose in the liquid fraction; X3 and G3 are furfural and HMF in the liquid fraction; and X4 and G4 are loss or other formation about xylose and glucose.
Fig. 3 e Relationship between pKa value and cellulose enzymatic hydrolysis. The CED of dilute sulfuric acid pretreated bagasse is 25.4%.
315
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relationship between pKa value of metal chloride and cellulose enzymatic digestibility of pretreated bagasse. It showed that the CED of bagasse pretreated by metal chloride Lewis acids was larger than that (only 25.4%) of bagasse pretreated by dilute sulfuric acid. It suggested bagasse was more vulnerable to cellulase after metal chloride pretreatment rather than sulfuric acid pretreatment. Moreover, Fe (III)-pretreated bagasse could release 96.7% glucose after 72 h hydrolysis by cellulase. In general, the cellulose enzymatic digestibility of bagasse pretreated increased with the decrease of pKa values of metal cations except Cr (III). The pKa value of Cr (III) was lower than Al (III). However, the Al (III)-pretreated bagasse was more readily hydrolyzed by cellulase than Cr (III)pretreated one. It suggested that some other factors might play an important role in the enzymatic hydrolysis of bagasse pretreated by metal chloride Lewis acids. For instance, different metal ions remained on the pretreated bagasse might have various effects on the cellulase hydrolysis. Chromium, as a heavy metal element, might make the cellulase denature and inactive, leading to a worse CED for enzymatic hydrolysis of Cr (III)-pretreated bagasse.
3.4. Kinetic study of cellulase hydrolysis of bagasse pretreated by metal chlorides As the discussion above, the factors that affected the enzymatic hydrolysis of pretreated bagasse were not simply owing to the pKa values. They might include enzymatic hydrolysis conditions, inhibitors as well as the complicated matric structural change of biomass after pretreatment. For instance, the modification of biomass structure would change the accessibility of cellulase to biomass, which is a crucial step for lignocellulosic biomass hydrolysis by cellulase. Usually, the absorption capability of cellulase on biomass was used to explain the accessibility. However, the absorption contained not only productive portion, but also unproductive one. Among the different kind of pretreated biomass, it was hard to verify the accessibility of cellulase because the components of different pretreated biomasses were difficult to explain the contribution of components to productive apart. There are some other factors that influence the accessibility of cellulase such as the crystallinity of cellulose, porosity and size of pretreated biomass. Hence, it is not appropriate to use the absorption of cellulase to determine the effect of the enzymatic hydrolysis of pretreated biomass. To further understand the performance of cellulase hydrolysis of pretreated biomass, it is vital to investigate the hydrolysis kinetics based on the relationship between hydrolysis product concentration and time. The enzymatic hydrolysis reaction of pretreated bagasse, like many other enzymatic hydrolysis reaction system, could be described as the following equation (2) [23]: ks
kcat
E þ S ! ES!E þ P 1
(2)
where cellulase (E) (g L ) was adsorbed on the active sites of the pretreated substrate (S) (g L1) to form the intermediate complexes (ES) (g L1), which continue to release free enzyme (E) and produce reducing sugar (P) (g L1).
Based on the equation (2), Michaelis and Menten inferred equation (3): v¼
d½P ½S ½S ¼ Vmax ¼ kcat E0 dt KM þ ½S KM þ ½S
(3)
where KM is experimentally defined as the concentration at which the rate of the enzyme reaction is half Vmax, which can be verified by substituting [S] ¼ KM into the MichaeliseMenten equation. However, some crucial assumptions underlie this equation. The assumptions are that the concentration of the substrate-bound enzyme and total enzyme maintain steady over time. Moreover, the assumption about the mechanism also involves no intermediate or product inhibition, and there is no allostericity or cooperativity. The cellulase hydrolysis of pretreated biomass is a complicated heterogeneous reactive system. The assumptions do not exit. Hence, the MichaeliseMenten equation has not been applied to the enzymatic hydrolysis of biomass. In order to taking diffusion limited factor into account, the equation (4) was proposed by Chrastil: n
P ¼ P∞ ½1 expð kcat E0 tÞ
(4)
where P and P∞ are the product concentration at time t and at equilibrium, respectively; kcat and E0 are the rate constant and initial enzyme concentration; while n represents the diffusion resistance constant. When the system is strongly diffusionlimited, n is small (high-resistance structures n ¼ 0.5e0.6). If the system could not take diffusion resistance into account, n tends to 1, which the reaction is of first order, approaching to the integrated form (5) of similar HenrieMichaeliseMenten equation (6) shown as follows: P ¼ P∞ ½1 expð kcat tÞ d½P ¼ kcat dt
P∞ P
(5)
(6)
In this study, the equation (6) was proposed as follow: 1
P ¼ P∞ ½1 expð ktÞIF
(7)
where P and P∞ were also the product concentration at time t and at equilibrium, respectively; the k was the constant that equaled to the term of kcatE0 described in the equation (4); the IF in equation (7) represented the inhibition factors. Fig. 4a here e showed the glucose concentration in supernatant varied with enzymatic hydrolysis time of bagasse pretreated by different metal chloride Lewis acids. It can be seen from the Fig. 4a that the glucose concentration tended to be constant after 48 h hydrolysis of pretreated bagasse. Therefore, the 72 h hydrolysis of pretreated bagasse could be regarded as the end of the enzymatic hydrolysis reaction, in which the glucose concentration was equivalent to the P∞. The all glucose concentration points dependent on the hydrolysis time were fixed with the equation (7) for the specific pretreated sample. Magnifying the points in red dash line (in web version) frame of Fig. 4a, a good linear relationship between glucose concentration and hydrolysis time could be found as shown in e Fig. 4b here. In this case, we could consider that the kinetics of hydrolysis before 2 h belonged to the zero order reaction, which meant the initial velocity of
316
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Fig. 5 e Relationship between CED, v0, k and IF.
due to the kinetic model making product concentration as the function of time at full range. Among the Lewis acid pretreated samples, the IFs were mainly between 1.35 and 1.89, much more than 1, suggesting that these pretreated samples have some extent inhibition to cellulase hydrolysis. With regard to dilute sulfuric acid pretreated bagasse, the IF was greater than 2.0, indicating the robust inhibition property than Lewis acid pretreatment. For all acid pretreated sample, the P∞, k and v0 rise with the increase of IF. Compared with cellulose enzymatic digestibility (CED) as shown in e Fig. 5 here, we could find when the IF declined from 2.14 to 1.35, the CED rise from 25.4% to 96.7%. In this case, the inhibitor factor might be used as an apparent parameter to compare the cellulase digestibility of biomass after different pretreatment. Fig. 4 e (a) Glucose concentration dependent on hydrolysis time from different pretreatment systems and (b) effect of pretreatment on the cellulase hydrolysis initial velocity of bagasse.
cellulase hydrolysis could be represented as the slope of line in the Fig. 4b. The parameters were summarized in Table 4. R2, larger than 0.99, indicated that the kinetic model for enzymatic hydrolysis of all the pretreated bagasse were significant. Hence, the kinetic model suited for the enzymatic hydrolysis at full time of bagasse pretreated by the metal chloride Lewis acids. Besides, the inhibition factors might include not only diffusion-limited, but also intermediate or product inhibition
Table 4 e Parameters of enzymatic hydrolysis kinetics for pretreated bagasse. Sample Fe(III) Al(III) Cr(III) Zn(II) Fe(II) H2SO4
P∞ g L1 9.50 ± 9.43 ± 7.88 ± 6.84 ± 5.34 ± 3.13 ±
0.04 0.03 0.06 0.13 0.15 0.25
k h1
IF
Adj. R2
v0 g L1 min1
0.191 0.111 0.106 0.102 0.0551 0.0226
1.35 1.51 1.56 1.67 1.89 2.14
0.9994 0.9998 0.9994 0.9973 0.9965 0.9998
0.0304 0.0288 0.0228 0.0193 0.0120 0.00396
4.
Conclusion
The enzymatic hydrolysis of bagasse is significantly affected by the metal chloride pretreatment of the raw materials. It was found that pKa values of metal chlorides are essential to pretreatment of bagasse, but not the only factor that affected the pretreatment due to the formation of complicate products from pretreatment and the accessibility of cellulase to substrates. An inhibition factor (IF) was proposed to describe the difference of metal chloride pretreatment. When inhibition factors decreased, cellulose enzymatic digestibility of pretreated bagasse, initial velocity of hydrolysis and k would increase, respectively.
Acknowledgments We acknowledge the financial support from the National Natural Science Foundation of China (No.31170549).
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Available online at www.sciencedirect.com
ScienceDirect http://www.elsevier.com/locate/biombioe
Preliminary exploration on pretreatment with metal chlorides and enzymatic hydrolysis of bagasse Liheng Chen, Rong Chen, Shiyu Fu* State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China
article info
abstract
Article history:
Converting biomass to fermentable sugar is the critical step in the biomass refinery.
Received 26 May 2014
Moreover, pretreatment of biomass plays an important role in improving the conversion of
Received in revised form
biomass to sugar. In this study, sugarcane bagasse was pretreated by metal chloride Lewis
24 September 2014
acids (0.1 mol L3 CrCl3, FeCl3, FeCl2, ZnCl2 and AlCl3 solution) for cellulase hydrolysis. The
Accepted 25 September 2014
effects of pretreatments on the yield, chemical components, and sequential cellulase hy-
Available online 16 October 2014
drolysis of pretreated bagasse were investigated. The results indicated that metal chlorides with different pKa values could efficiently remove the hemicellulose in bagasse during
Keywords:
pretreatment. Furthermore, an inhibition factor (IF) quantitatively reflecting difficulty of
Sugarcane bagasse
cellulase hydrolysis was proposed. The low IF means the facile cellulase hydrolysis. The IF
pKa value
of Fe (III)-pretreated bagasse could decrease to 1.35. In this case, the enzymatic digestibility
Metal chloride Lewis acid
of bagasse approached to 100%.
Pretreatment
© 2014 Elsevier Ltd. All rights reserved.
Cellulase hydrolysis
1.
Introduction
To pursue alternatives to fossil energies is an indispensable trend, as the fossil energies suffer from the limited source and deteriorating environment [1,2]. Biofuels such as bio-alcohols, regarded as environmentally friendly alternatives, have been exploited extensively [3]. Lignocellulosic bioconversion is a competitive way to produce bioethanol because of its mild operating condition and the absence of by-products [4]. However, lignocellulose, feedstock of the process, has a rigid matrix structure comprised of cellulose, hemicellulose and lignin [5]. The presence of lignin and hemicellulose makes lignocellulosic bioconversion, especially the enzymatic hydrolysis of cellulose, hard to be implemented [6,7]. Generally, * Corresponding author. Tel.: þ86 20 87113940; fax: þ86 20 22236078. E-mail address: [email protected] (S. Fu). http://dx.doi.org/10.1016/j.biombioe.2014.09.026 0961-9534/© 2014 Elsevier Ltd. All rights reserved.
the factors that have been identified to affect the hydrolysis of cellulose include porosity (accessible surface area) of the waste materials, cellulose fiber crystallinity, and lignin and hemicellulose content [8]. The removal of lignin and hemicellulose from biomass, reduction of cellulose crystallinity and augment of porosity in pretreatment processes can significantly improve the enzymatic hydrolysis of biomass. Hence, pretreatment prior to enzymatic hydrolysis is imperative for lignocellulosic feedstock. Variety of biomass pretreatments have been reported to promote the lignocellulosic bioconversion and cut down the total costs. The pretreatment is usually performed by steam explosion, liquid hot water, dilute acid, inorganic salt, lime and ammonia [9]. Each pretreatment method has its own advantages and disadvantages
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for specific lignocellulosic feedstock [9]. In all cases, pretreatment, which could effectively remove hemicellulose in biomass, is crucial to enzymatic hydrolysis of lignocellulose. However, a complicated factor is that different biomass applied to the same pretreatment process will present different patterns of hemicellulose or lignin removal and of enzymatic digestibility, even under the same conditions [10]. In this study, sugarcane bagasse was pretreated by metal chloride Lewis acids for cellulase hydrolysis. With regard to the feedstock, sugarcane bagasse is a typical agricultural residue, derived from sugarcane after juice extraction [11]. In order to make full use of bagasse, it is essential to pursue a promising route such as bioconversion [12]. For pretreatment methods, metal chlorides have been applied to some biomass species, such as corn stover and waste fiber, which were reported to have a great effect on the sequent enzymatic hydrolysis of biomass [13e15]. Metal chlorides are particularly attractive as lignocellulose pretreatment agents, because they are less corrosive than inorganic acids and can be recycled [13]. Moreover, these metal chlorides were performed on the conversion of bagasse to sugar could not only expand the use of bagasse, but explore the suitability of this pretreatment process. Since high efficiency and economic feasibility of pretreatment is vital for large scale commercialization of biofuels and value added bio-chemicals, the kinetics of pretreatment for lignocellulosic bioconversion need to study to get an optimal condition. However, little information about the kinetics of enzymatic hydrolysis of lignocellulose pretreated by metal chlorides was reported except the effects of metal chlorides on biomass pretreatment [16]. In the recent years, the MichaeliseMenten equation has been widely used to study kinetics of enzymes performed on various substrates [17,18]. Nevertheless, it is not suitable for cellulase hydrolysis of lignocellulosic biomass, owing to the complexity of the enzymatic hydrolysis of biomass which is heterogeneous insoluble substrates [17]. Herein, we would investigate the effects of metal chloride Lewis acids pretreatment on the bagasse enzymatic hydrolysis, as well as explore the kinetics for further understanding the mechanism of these pretreatment.
2.
Materials and methods
2.1.
Materials
The feedstock, sugarcane bagasse, was obtained from Guangxi Guitang Group, Guangxi province, China. The sugarcane (Saccharum spp. hybrids) used in this sugar mill was cultivated in Guigang (Guangxi province, China) and harvested at an age of (10e11) months by harvester. The sugarcane was pressed through rollers to abstract the juice. The solid residues, known as bagasse, were air-dried and used in this study after depithing. They were milled by micro plant grinding machine (FZ102, Tianjin Taisite Instrument Co. China), collected through 10-mesh screen and then air-dried for further use. The mass fractions of glucan, xylan, lignin and ash in air-dried sugarcane bagasse were (36.02 ± 0.31) %, (19.41 ± 0.07) %, (24.20 ± 1.60) % and (0.55 ± 0.03) %,
respectively. Chemicals used in this paper were of analytical reagent grade. All experiments were performed in duplicate under the same conditions, and average values were reported.
2.2.
Pretreatment
Pretreatment was performed in a high pressure reactor (4530 series, Parr Co., US) with a total volume of 1 L. The bagasse of 30 g dry weight was loaded in the reactor, and mixed with the metal chloride solution. The total volume of liquid was 300 cm3. The initial concentration of metal chloride (CrCl3, FeCl2, FeCl3, ZnCl2 and AlCl3) for the pretreatment was 0.1 mol L1. In addition, dilute sulfuric acid pretreatment was carried out in this study as a control method to compare with metal chloride pretreatment. The sulfuric acid (about 0.4 g) was added until the pH value equaled to solution of ferric chloride pretreatment. After complete agitation, the pH value of solution was detected by DELTA 320 pH Meter. The reactants were initially at room temperature, and then heated to 170 C for 30 min. The reactor was immediately removed from the heating jacket and allowed to cool down when the pretreatment was finished. The pretreated bagasse was then separated by filtration. The liquid part of pretreatment was analyzed by pH meter, ion chromatography (IC) and highpressure liquid chromatography (HPLC) to determine pH values, the concentration of glucose, xylose, 5hydroxymethylfurfural (HMF) and furfural, respectively. The solid portion rinsed with deionic water was used for the cellulase hydrolysis. Meanwhile, composites in the pretreated solid were analyzed.
2.3.
Enzymatic hydrolysis
Pretreated bagasse of 1 g dry weight was hydrolyzed in the 100 mL flask by cellulase (Celluclast 1.5 L) with 20 Filter Paper Unit (FPU) per 1 g substrate and b-glucosidase (Novozyme 188) with 25 cellobiase units (CBU) per 1 g substrate. The cellulase hydrolysis of reaction mixture (50 cm3) was carried out in a HAc/NaAc buffer (0.05 mol L1, pH ¼ 4.8) on a rotary shaker (50 C, 2.5 Hz). Aliquots of 0.1 cm3 were taken at different time points, kept in boiling water for 1 min to inactivate the cellulase, and then centrifuged to remove water-insoluble solids. The supernatant of samples was analyzed by the glucose oxidaseperoxidase method (GOPM) for glucose content [19]. The cellulose enzymatic digestibility (CED) of pretreated bagasse was described as follows: CED ¼
2.4.
0:9 Glucose Mass 100% Substrate Mass Mass Fractrion of Glucan
(1)
Analysis methods
The components of treated fibers were determined according to the National Renewable Energy Laboratory (NREL, Golden, CO) analytical methods for biomass [20]. All liquid fractions for analysis by IC were diluted appropriately with the ultrapure water and then filtered by a 0.22 mm filter. The concentration of glucose and xylose were determined by IC system (Dionex ICS-3000) with a CarboPac PA20 column at 30 C. The quantification of HMF and furfural in the pretreatment
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solution were carried out by HPLC system with C18 column at 30 C based on a diode array detector (DAD). The eluents containing methanol (30%, volume basis) and water (70%, volume basis) flew at a rate of 1.0 cm3 min1.
3.
Results and discussion
3.1. Hydrolysis reaction of metal chlorides in water during pretreatment Generally, the acid strength plays an important role in acid pretreatments of lignocellulose [21]. Among metal chlorides pretreatment, the pKa values based on the acid dissociation constant were determined to indicate their acid strength [16]. Table 1 here e summarizes the pKa values of different metal chlorides in water at 25 C [22]. The lower pKa value indicated the more robust acidic character of the metal ion in the solution. The data in Table 1 showed that Fe (III), which pKa value is 2.73, forms stronger acids in aqueous solution than other cations. The pH values were measured for all the solutions (metal chloride solution mixed with bagasse) prior to pretreatment and after pretreatment. Fig. 1 here e was described as the relationship between initial and final pH values of liquid portion during different pretreatments. The red dash line (in web version) indicated the equivalent value of initial and final pH. When the data were located upon the dash line, it means that the final pH value was larger than the initial one. On the contrary, the final pH value would be less than the initial one. As we see from Fig. 1, pH values of liquid fraction in all pretreatment systems except dilute sulfuric acid pretreatment decreased. These results demonstrated that metal chlorides underwent hydrolysis to release more hydrogen ions during pretreatment as weak electrolyte. Hence, the ongoing releasing hydrogen ions and the probable produce of acetic acid from hemicellulose cause inevitably the decrease of the pH values of liquor after metal chloride pretreatments. Nevertheless, the sulfuric acid is a stronger electrolyte than acetic acid, which is the weak one. During the dilute sulfuric acid pretreatment, the sulfuric acid hydrolyzed entirely firstly. The exhaustion of hydrogen ions was much more than the produce from acetic acid hydrolysis, which resulted in the pH value rising from 1.6 to 3.82. Compared with the inorganic acids of strong electrolyte, metal chloride Lewis acids could provide steadier reactive environment and be less corrosive as well.
3.2. Component change of sugarcane bagasse after different metal chloride pretreatment It is essential to detect the component change of lignocellulosic material during pretreatment. The main components
Table 1 e The pKa value at 25 C. Methods
25 C pKa
Fe(III)
Cr(III)
Al(III)
Fe(II)
Zn(II)
2.74
4.01
5.03
6.8
6.31
Fig. 1 e Initial and final pH values of liquid portion during different pretreatment systems.
(glucan, xylan and Klason lignin) of sugarcane bagasse (SB) pretreated by different metal chlorides and dilute sulfuric acid were summarized in e Table 2 here. In general, the mass fractions of glucan and lignin in pretreated bagasse increased in comparison to those in raw bagasse, and the mass fraction of xylan in bagasse after pretreatment decreased significantly, which made the bagasse suffer from numeric mass loss. For instance, the largest remained solid mass of bagasse pretreated by dilute sulfuric acid was only 74.5%, and the least remained solid mass could reduce to 54.0% after chrome chloride pretreatment. The reason for mass loss of bagasse was mainly that the carbohydrates in bagasse degraded during pretreatment in the acidic environment. The extent of carbohydrate degradation could be also detected via analysis of pretreatment effluent. Table 3 here e outlined the components of pretreatment effluent. Numerous monosaccharides such as xylose and glucose were released, even further degraded to furfural and 5-hydroxymethylfurfural (HMF) as shown in Table 3. The greatest concentration (13.5 g L1 and 7.55 g L1) of xylose and glucose in pretreatment effluent could be attained during Cr (III) pretreatment. However, pretreatment by Fe (III) could produce more furfural, which reached 5.11 g L1, greater than that by Cr (III). It suggested that xylose during Fe (III) pretreatment was conversed to furfural more easily.
Table 2 e Mass fractions of components in pretreated sugarcane bagasse (%). Sample
Solid remain
Raw SB Fe(III) Cr(III) Al(III) Fe(II) Zn(II) H2SO4
e 58.0 54.0 70.4 71.3 72.2 74.5
Glucan 36.0 46.6 43.7 50.2 51.3 50.9 49.9
± 0.3 ± 2.3 ± 1.6 ± 1.4 ± 1.3 ± 0.9 ± 0.7
Xylan 19.4 ± 2.15 ± 3.33 ± 9.06 ± 12.8 ± 14.7 ± 16.7 ±
0.1 0.78 0.02 0.9 0.7 1.2 0.5
Lignin 24.2 41.3 44.7 35.0 33.3 33.7 32.3
± 1.6 ± 1.3 ± 0.6 ± 1.1 ± 0.6 ± 1.2 ± 0.9
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Table 3 e Components of pretreatment effluent. Concentration (g L1)
Method Xylose Fe(III) Cr(III) Al(III) Fe(II) Zn(II) H2SO4
11.3 ± 13.5 ± 10.2 ± 6.31 ± 3.61 ± 3.40 ±
0.3 0.1 0.1 0.08 0.23 0.10
Glucose
Furfural
6.77 ± 0.12 7.55 ± 0.24 0.736 ± 0.031 1.25 ± 0.04 0.357 ± 0.009 0.325 ± 0.013
Fig. 2a and b here e illustrated the existing forms of xylan and glucan in bagasse after different pretreatment and its relationship with pKa values of metal chlorides. The carbohydrates such as xylan and glucan underwent various extent degradations during pretreatment by metal chlorides of different pKa values. Especially, the xylan was more readily to
5.11 1.88 1.70 3.72 3.46 0.870
± 0.15 ± 0.47 ± 0.10 ± 0.10 ± 0.05 ± 0.002
HMF 0.752 0.667 0.816 0.268 0.252 0.069
± 0.030 ± 0.003 ± 0.058 ± 0.007 ± 0.005 ± 0.022
be destroyed among the pretreatment systems. In general, the xylan remained in bagasse pretreated by metal chlorides improved with the increase of pKa values, but less than that by dilute sulfuric acid pretreatment (Fig. 2a). The capability of pretreatment systems about degrading xylan was as follow: Fe (III) > Cr (III) > Al (III) > Fe (II) > Zn (II) > H2SO4 (Fig. 2a). The xylan was almost removed entirely in the Fe (III) and Cr (III) pretreated bagasse, their removal rates could reach 93.6% and 90.7%, respectively. Furthermore, glucan in these two pretreated bagasse suffered from some extent degradation, where the recovery of glucan decreased to 75.1% and 65.6%, respectively (Fig. 2b). However, other pretreatment systems could hardly degrade glucan. These results might result from the lower pKa values of Fe (III) and Cr (III) than others. In addition, the recovery of lignin in pretreated bagasse was approximately 100%, which indicated that all these pretreatment systems did not have delignification effect.
3.3. The pKa values of metal chlorides on the cellulose enzymatic digestibility of pretreated bagasse The pKa values of metal chlorides had been proved to have great influence on the bagasse pretreatment by metal chlorides. In this part, we tried to find out how the pKa value of metal chloride Lewis acids affected the sequential enzymatic hydrolysis of pretreated bagasse. Fig. 3 here e depicted the
Fig. 2 e Existing forms of xylan (a) and glucan (b) in bagasse after being pretreated with metal chloride and dilute sulfuric acid. X1 and G1 are the remaining xylan and glucan in the residue; X2 and G2 are monomeric xylose and glucose in the liquid fraction; X3 and G3 are furfural and HMF in the liquid fraction; and X4 and G4 are loss or other formation about xylose and glucose.
Fig. 3 e Relationship between pKa value and cellulose enzymatic hydrolysis. The CED of dilute sulfuric acid pretreated bagasse is 25.4%.
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relationship between pKa value of metal chloride and cellulose enzymatic digestibility of pretreated bagasse. It showed that the CED of bagasse pretreated by metal chloride Lewis acids was larger than that (only 25.4%) of bagasse pretreated by dilute sulfuric acid. It suggested bagasse was more vulnerable to cellulase after metal chloride pretreatment rather than sulfuric acid pretreatment. Moreover, Fe (III)-pretreated bagasse could release 96.7% glucose after 72 h hydrolysis by cellulase. In general, the cellulose enzymatic digestibility of bagasse pretreated increased with the decrease of pKa values of metal cations except Cr (III). The pKa value of Cr (III) was lower than Al (III). However, the Al (III)-pretreated bagasse was more readily hydrolyzed by cellulase than Cr (III)pretreated one. It suggested that some other factors might play an important role in the enzymatic hydrolysis of bagasse pretreated by metal chloride Lewis acids. For instance, different metal ions remained on the pretreated bagasse might have various effects on the cellulase hydrolysis. Chromium, as a heavy metal element, might make the cellulase denature and inactive, leading to a worse CED for enzymatic hydrolysis of Cr (III)-pretreated bagasse.
3.4. Kinetic study of cellulase hydrolysis of bagasse pretreated by metal chlorides As the discussion above, the factors that affected the enzymatic hydrolysis of pretreated bagasse were not simply owing to the pKa values. They might include enzymatic hydrolysis conditions, inhibitors as well as the complicated matric structural change of biomass after pretreatment. For instance, the modification of biomass structure would change the accessibility of cellulase to biomass, which is a crucial step for lignocellulosic biomass hydrolysis by cellulase. Usually, the absorption capability of cellulase on biomass was used to explain the accessibility. However, the absorption contained not only productive portion, but also unproductive one. Among the different kind of pretreated biomass, it was hard to verify the accessibility of cellulase because the components of different pretreated biomasses were difficult to explain the contribution of components to productive apart. There are some other factors that influence the accessibility of cellulase such as the crystallinity of cellulose, porosity and size of pretreated biomass. Hence, it is not appropriate to use the absorption of cellulase to determine the effect of the enzymatic hydrolysis of pretreated biomass. To further understand the performance of cellulase hydrolysis of pretreated biomass, it is vital to investigate the hydrolysis kinetics based on the relationship between hydrolysis product concentration and time. The enzymatic hydrolysis reaction of pretreated bagasse, like many other enzymatic hydrolysis reaction system, could be described as the following equation (2) [23]: ks
kcat
E þ S ! ES!E þ P 1
(2)
where cellulase (E) (g L ) was adsorbed on the active sites of the pretreated substrate (S) (g L1) to form the intermediate complexes (ES) (g L1), which continue to release free enzyme (E) and produce reducing sugar (P) (g L1).
Based on the equation (2), Michaelis and Menten inferred equation (3): v¼
d½P ½S ½S ¼ Vmax ¼ kcat E0 dt KM þ ½S KM þ ½S
(3)
where KM is experimentally defined as the concentration at which the rate of the enzyme reaction is half Vmax, which can be verified by substituting [S] ¼ KM into the MichaeliseMenten equation. However, some crucial assumptions underlie this equation. The assumptions are that the concentration of the substrate-bound enzyme and total enzyme maintain steady over time. Moreover, the assumption about the mechanism also involves no intermediate or product inhibition, and there is no allostericity or cooperativity. The cellulase hydrolysis of pretreated biomass is a complicated heterogeneous reactive system. The assumptions do not exit. Hence, the MichaeliseMenten equation has not been applied to the enzymatic hydrolysis of biomass. In order to taking diffusion limited factor into account, the equation (4) was proposed by Chrastil: n
P ¼ P∞ ½1 expð kcat E0 tÞ
(4)
where P and P∞ are the product concentration at time t and at equilibrium, respectively; kcat and E0 are the rate constant and initial enzyme concentration; while n represents the diffusion resistance constant. When the system is strongly diffusionlimited, n is small (high-resistance structures n ¼ 0.5e0.6). If the system could not take diffusion resistance into account, n tends to 1, which the reaction is of first order, approaching to the integrated form (5) of similar HenrieMichaeliseMenten equation (6) shown as follows: P ¼ P∞ ½1 expð kcat tÞ d½P ¼ kcat dt
P∞ P
(5)
(6)
In this study, the equation (6) was proposed as follow: 1
P ¼ P∞ ½1 expð ktÞIF
(7)
where P and P∞ were also the product concentration at time t and at equilibrium, respectively; the k was the constant that equaled to the term of kcatE0 described in the equation (4); the IF in equation (7) represented the inhibition factors. Fig. 4a here e showed the glucose concentration in supernatant varied with enzymatic hydrolysis time of bagasse pretreated by different metal chloride Lewis acids. It can be seen from the Fig. 4a that the glucose concentration tended to be constant after 48 h hydrolysis of pretreated bagasse. Therefore, the 72 h hydrolysis of pretreated bagasse could be regarded as the end of the enzymatic hydrolysis reaction, in which the glucose concentration was equivalent to the P∞. The all glucose concentration points dependent on the hydrolysis time were fixed with the equation (7) for the specific pretreated sample. Magnifying the points in red dash line (in web version) frame of Fig. 4a, a good linear relationship between glucose concentration and hydrolysis time could be found as shown in e Fig. 4b here. In this case, we could consider that the kinetics of hydrolysis before 2 h belonged to the zero order reaction, which meant the initial velocity of
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Fig. 5 e Relationship between CED, v0, k and IF.
due to the kinetic model making product concentration as the function of time at full range. Among the Lewis acid pretreated samples, the IFs were mainly between 1.35 and 1.89, much more than 1, suggesting that these pretreated samples have some extent inhibition to cellulase hydrolysis. With regard to dilute sulfuric acid pretreated bagasse, the IF was greater than 2.0, indicating the robust inhibition property than Lewis acid pretreatment. For all acid pretreated sample, the P∞, k and v0 rise with the increase of IF. Compared with cellulose enzymatic digestibility (CED) as shown in e Fig. 5 here, we could find when the IF declined from 2.14 to 1.35, the CED rise from 25.4% to 96.7%. In this case, the inhibitor factor might be used as an apparent parameter to compare the cellulase digestibility of biomass after different pretreatment. Fig. 4 e (a) Glucose concentration dependent on hydrolysis time from different pretreatment systems and (b) effect of pretreatment on the cellulase hydrolysis initial velocity of bagasse.
cellulase hydrolysis could be represented as the slope of line in the Fig. 4b. The parameters were summarized in Table 4. R2, larger than 0.99, indicated that the kinetic model for enzymatic hydrolysis of all the pretreated bagasse were significant. Hence, the kinetic model suited for the enzymatic hydrolysis at full time of bagasse pretreated by the metal chloride Lewis acids. Besides, the inhibition factors might include not only diffusion-limited, but also intermediate or product inhibition
Table 4 e Parameters of enzymatic hydrolysis kinetics for pretreated bagasse. Sample Fe(III) Al(III) Cr(III) Zn(II) Fe(II) H2SO4
P∞ g L1 9.50 ± 9.43 ± 7.88 ± 6.84 ± 5.34 ± 3.13 ±
0.04 0.03 0.06 0.13 0.15 0.25
k h1
IF
Adj. R2
v0 g L1 min1
0.191 0.111 0.106 0.102 0.0551 0.0226
1.35 1.51 1.56 1.67 1.89 2.14
0.9994 0.9998 0.9994 0.9973 0.9965 0.9998
0.0304 0.0288 0.0228 0.0193 0.0120 0.00396
4.
Conclusion
The enzymatic hydrolysis of bagasse is significantly affected by the metal chloride pretreatment of the raw materials. It was found that pKa values of metal chlorides are essential to pretreatment of bagasse, but not the only factor that affected the pretreatment due to the formation of complicate products from pretreatment and the accessibility of cellulase to substrates. An inhibition factor (IF) was proposed to describe the difference of metal chloride pretreatment. When inhibition factors decreased, cellulose enzymatic digestibility of pretreated bagasse, initial velocity of hydrolysis and k would increase, respectively.
Acknowledgments We acknowledge the financial support from the National Natural Science Foundation of China (No.31170549).
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